A case of familial hypocalciuric hypercalcemia type 1 due to CASR p.Pro55Leu mutation

Background Familial hypocalciuric hypercalcemia (FHH) is a rare autosomal dominant disease, which requires differential diagnosis from relatively common primary hyperparathyroidism (PHPT) in order to avoid unnecessary surgery. Case presentation A 16-year-old female had been followed by the department of psychosomatic medicine at our institution. Throughout the follow-up period, her plasma calcium levels were high, plasma Pi levels were relatively low, and plasma intact PTH was relatively high. She was referred to our department to determine the cause of her hypercalcemia. Her 24 h urinary calcium excretion was as low as 100 mg/day, and calcium creatinine clearance ratio was below 0.01. Moreover, she had a family history of hypercalcemia (proband, her brother, and her father). The genetic testing for her family revealed that she, her brother, and her father were definitively diagnosed with FHH type 1 due to the heterozygous calcium-sensing receptor mutation (NM_00388:4:c.164C > T:p.Pro55Leu). Conclusion We experienced a 16-year-old female with FHH, in whom genetic testing identified the heterozygous calcium-sensing receptor mutation (NM_00388:4:c.164C > T:p.Pro55Leu) as pathogenic, permitting a definitive diagnosis of FHH type 1. The genetic testing for calcium sensing receptor is beneficial to distinguish asymptomatic primary hyperparathyroidism from FHH.


Background
Hypercalcemia is a condition causing disorders in multiple organs, including bone, brain, and heart [1]. The causes of hypercalcemia include hyperparathyroidism, cancer, tuberculosis, immobility, and inappropriate vitamin D supplementation. Among these, primary hyperparathyroidism (PHPT) is a relatively common disease (incidence rate;1 in 1,000) and often requires parathyroid gland resection [2]. Familial hypocalciuric hypercalcemia (FHH) is a rare autosomal dominant disease (incidence rate; 1 in 78,000) that requires, in general, no treatment [2,3]. Thus, differential diagnosis of FHH from PHPT is critical to avoid unnecessary surgery.
To date, FHH-related mutations have been identified in genes related to the calcium sensing system that regulates calcium homeostasis [2]. The calcium-sensing receptor, CASR, is encoded by the CASR gene and belongs to a G protein-coupled receptor family that regulates parathyroid hormone (PTH) secretion, vitamin D synthesis, and calcium absorption and resorption [2,5]. G-protein α11 (GNA11) is encoded by the GNA11 gene and mediates CaSR signaling [2,5]. Adaptor-related protein complex 2 (AP-2) is a membraneassociated heterotetramer complex and composed of two large chain, medium chain, and a small chain. The adaptor-related protein complex 2 sigma one subunit encoded by the AP2S1 gene binds to CASR and facilitates CaSR internalization [2,5]. Loss of function mutations in the CASR, GNA11, or AP2S1 genes cause FHH type 1 (FHH1), FHH type 2 (FHH2), and FHH type 3 (FHH3), respectively [1,4]. Approximately 60% of FHH patients have mutations in the CASR gene [2].
Here we report a case of FHH1 in which genetic testing identified the heterozygous CASR mutation p.Pro55Leu, which permitted the definitive diagnosis of the disease. In this case with no symptom, family history and genetic test for CASR was beneficial for the diagnosis of FHH type1.

Case presentation
A 16-year-old female had been followed by the department of psychosomatic medicine at our institution for borderline personality disorder. Throughout the followup period, her plasma calcium levels were high, plasma Pi levels were relatively low, and plasma intact PTH (PTHi) was relatively high (Fig. 1).
Since her brother (II-1) and father (I-1) had hypercalcemia ( Fig. 2A), we proposed that she receive a genetic test to diagnose with FHH and she and her family agreed with our proposal. Whole exon sequencing was performed by fragmentation of DNA, enrichment of exonic regions using capture-based hybridization, PCR amplification, and rapid sequencing using a next-generation sequencer in Kazusa DNA laboratory. Exon sequencing of hereditary hypercalcemia-related genes (e.g., MEN1, CDKN1B, RET, CASR, GNA11, AP2S1, CDC73, GCM2) was performed and a pathogenic CASR mutation (CASR: NM_000388.4: exon2: c.164C > T: p. Pro55Leu) was noted in her father (I-1), brother (II-1), and the proband (II-3) (Fig. 2B), but no mutation in GNA11 and AP2S1 was identified. Based on the American College of Medical Genetics and Genomics Standards and Guidelines, the present variant was classified as pathogenic due to PS1 + PS3 + PM1 + PM2 [7]. She was then positively diagnosed as FHH1. As she was diagnosed with FHH, we followed up her with no medication. She and her family were relieved to hear that she needed no surgical treatment. Her plasma Ca 2+ and Pi levels were almost unchanged for 18 months (Fig. 1).

Discussion and conclusions
We report here a case of hypercalcemia with family history and low CCCR with hypovitaminosis D, in which genetic testing permitted definitive diagnosis of FHH1.
FHH is a very rare benign inherited condition that typically does not require parathyroidectomy. Patients with FHH usually have no symptoms and are often diagnosed by chance during routine blood examination. Weakness, fatigue, issues with concentration, constipation, polyuria, headache, and polydipsia have been reported by some people with FHH. The calcimimetic drugs are sometimes effective to improve hypercalcemic symptoms such as muscle aches, anorexia, polydipsia and constipation [2]. Rarely, people with this disorder experience pancreatitis or a chondrocalcinosis [8]. Although she had no past-history of pancreatitis and bone fractures, we may follow up her risk of pancreatitis due to hypercalcemia through her life [8].
In the current study, a heterozygous CASR mutation p.Pro55Leu was found to be pathogenic in the patient's father (I-1), brother (II-1) and the proband (II-3). This mutation has previously been found in several individuals with FHH including one Japanese [2,[8][9][10][11][12][13][14][15][16]; it was classified as pathogenic in the ClinVar database (Variation ID: 279,731). The mutation is localized in the N-terminal extracellular domain (ECD) of CaSR, where ligand binding occurs; it causes a rightward shift of the PTH dose-response curve in response to extracellular calcium [11,16]. Consistent with available information regarding the mutation, the patient showed relatively high PTHi despite the high plasma calcium level (Table 1 and Fig. 1).
In conclusion, we report a case of FHH1 due to CASR p.Pro55Leu mutation, in which only genetic testing

Availability of data and materials
Clinical data from the corresponding author will be available upon request.